The present invention relates to (i) enzyme-containing polymeric sensors, and, especially, to sensors in which an enzyme or enzymes and an indicator compound or indicator compounds are incorporated within a common or single polymer matrix and to (ii) substrate-containing polymeric sensors, and especially, to sensors in which a substrate compound or substrate compounds and an indicator compound or indicator compounds are incorporated within a common or single polymer matrix.
Enzymatic proteins are remarkable natural catalysts that selectively catalyze many reactions under relatively mild reaction conditions. Enzymes also offer the potential to perform sterio- and regio-selective reactions not readily accomplished with conventional chemistry. As used herein, the term xe2x80x9cenzymexe2x80x9d refers generally to proteins that catalyze biochemical reactions. These xe2x80x9cbiopolymersxe2x80x9d include amide-linked amino acids and typically have molecular weights of 5,000 or greater. A compound for which a particular enzyme catalyzes a reaction is typically referred to as a xe2x80x9csubstratexe2x80x9d of the enzyme.
In general, six classes or types of enzymes (as classified by the type of reaction that is catalyzed) are recognized. Enzymes catalyzing reduction/oxidation or redox reactions are referred to generally as EC 1 (Enzyme Class 1) Oxidoreductases. Enzymes catalyzing the transfer of specific radicals or groups are referred to generally as EC 2 Transferases. Enzymes catalyzing hydrolysis are referred to generally as EC 3 hydrolases. Enzymes catalyzing removal from or addition to a substrate of specific chemical groups are referred to generally as EC 4 Lyases. Enzymes catalyzing isomeration are referred to generally as EC 5 Isomerases. Enzymes catalyzing combination or binding together of substrate units are referred to generally as EC 6 Ligases.
Enzymes have been known since the early 1960""s to be useful tools for detecting the presence of chemical species. Rogers, K. R., Biosensors Bioelectronics, 10, 533 (1995). Generally all enzymatic biosensors function by one of two methods. The enzyme either converts an undetectable compound of interest into another or series of compounds which can be detected with a chemical-based sensor or the enzyme is inhibited by the presence of the compound of interest and the enzyme inhibition is linked to a measurable quantity.
Enzymatic biosensors have been designed to detect a variety of different compounds such as glucose, creatinine, urea, and cholinesterase inhibitors. Parente, A. H., Marques, E. T. Jr., Appl. Biochem. Biotechnol. 37, 3, 267 (1992); Yang, S., Atanasov, P., Wilkins, E., Ann. Biomed. Eng., 23, 6, 833 (1995). U.S. Pat. No. 5,858,186 describes a urea-based biosensor in which substrate hydrolysis is monitored with a pH electrode. U.S. Pat. Nos. 5,945,343 and 5,958,786 describe enzyme-based sensors in which a fluorophere is immobilized in a first polymer layer and an enzyme is separately immobilized in a second polymer layer. The fluorophere layer fluoresces in the presence of ammonia, which is enzymatically produced from urea and creatinine (respectively, with respect to U.S. Pat. Nos. 5,945,343 and 5,958,786). In addition, U.S. Pat. No. 4,324,858 describes the immobilization of cholinesterase within a porous, dry material for the colormetric detection of organophosphorus pesticides and nerve agents. U.S. Pat. No. 4,525,704 describes the use of cholinesterases and electrical currents in detecting toxic gases.
Independent of the use thereof, enzyme-based biosensors are often limited in practical application by a number of factors. For example, the process of immobilizing the enzymes using highly specialized synthesis protocols is often expensive and time consuming. Moreover, the sensor often requires specialized electrical equipment to be used in conjunction with the immobilized enzyme, such as a pH meter or oxygen electrode. Turner, A. P. F., Sensors Actuators, 17, 433 (1989). The shelf-life, thermal stability, and reusability of enzymatic sensors is often problematic for practical application of the technology. Also, many enzyme-based sensors do not exhibit sufficient sensitivity toward the target compound to monitor the compound over a relevant concentration range. Evtugyn, G. A., Budnikov, H. C., Kolskaya, Talanta, 46, 465 (1998).
It is very desirable to develop improved enzyme-based sensors that reduce or eliminate the problems with current enzyme-based sensors.
In one aspect, the present invention provides a sensor for detecting the presence of at least one analyte. The sensor includes at least one enzyme that is selected to either (i) catalyze a reaction of the analyte to chemically convert the analyte to a product compound or (ii) be inhibited by the analyte in the presence of a substrate compound. The sensor also includes at least one indicator compound selected to produce a measurable change of state as a result of the interaction of the analyte and the enzyme. Each of the enzyme and the indicator compound are incorporated within a single polymer. Any polymer capable of retaining an enzyme and indicator compound that provides the incorporated/immobilized species with access to substrate, analyte, and/or the reaction products and is capable of undergoing a physical property change induced by the indicator compound is suitable for use in the present invention. The enzyme and/or the indicator compound can be added to a preexisting polymer or the polymer can be synthesized in the presence of the enzyme and/or the indicator compound to incorporate the enzyme and/or the indicator compound in the polymer.
In general, in the case that the analyte is converted to a product compound in a reaction catalyzed by the enzyme (that is, case (i), in which the analyte is a substrate of the enzyme), the indicator compound may be sensitive to the presence of the product compound and will produce a measurable change of state in the presence of the product compound.
In the case that the analyte inhibits the enzyme (case (ii), in which the analyte is not a substrate of the enzyme) the indicator compound may be sensitive to a product of the reaction of a substrate compound of the enzyme. In case (ii), the substrate compound is preferably added to the environment. If the indicator compound produces no change of state or a change of state that is less in degree than expected with an uninhibited enzyme, it is an indication of the presence of the analyte. Thus, enzyme inhibitors are detected by first incubating the enzyme and indicator compound-containing polymer within a sample of questionable nature. A xe2x80x9cdeveloping solutionxe2x80x9d containing the enzyme""s substrate is later applied. The absence of a state transition yields a positive indication of inhibition and the presence of the target compound.
The indicator compound may, for example, be a dye that undergoes an observable change of state (for example, a change in optical properties/color) as a result of the interaction of the enzyme and the analyte. Preferably, such a dye changes optical properties in a manner that is visible to the human eye. The indicator compound may be chemically bonded to the polymer or physically entrapped therein. An example of a suitable polymer for use in the present invention is polyurethane.
The enzyme is preferably chemically bonded to the polymer, but can also be physically entrapped within the polymer. More preferably, the enzyme is covalently bonded to the polymer. In one embodiment, the enzyme is a hydrolase enzyme and the indicator compound is a dye that changes color as a function of pH. Examples of suitable hydrolase enzymes include, but are not limited to, a lipase, a phosphatase, an amylase, a cellulase, a protease a peptidase, a urease or a deaminase. Specific examples of suitable hydrolases include, but are not limited to, organophosphorus hydrolase, organophosphorus acid anhydrolase, urease, butyrylcholinesterase or acetylcholinesterase. One or a plurality of types of enzymes can be incorporated within the polymer to detect one or a plurality of analytes. Examples of pH-sensitive dyes suitable for use with such enzymes include, but are not limited to, Brilliant green, crystal violet, methyl green, picric acid, Eosin Y, thymol blue, xylonel blue, Eosin B, cresol red, methyl yellow, ethyl orange, bromocresol green, Alizarin Red, bromomethyl blue, bromocresol purple, phenol red, and chlorophenol red.
In general, any enzyme and indicator compound (or compounds) combination that interact to produce a measurable change of state can be used in the polymers of the present invention. For example, 2,2xe2x80x2Dipyrdyl indicator dye changes color in the presence of Fe2+. Thus, any enzyme using Fe2+ as a substrate or producing Fe2+ in a product can be used in conjunction therewith. Likewise, Calcium Green-1 and Fluo-3 AM (available from Molecular Probes, Inc.) undergo a color change in the presence of Ca2+. Enzymes using Ca2+ using or producing Ca2+ during catalysis can be used with these dyes. For example, calmodulin is a calcium-binding protein exhibiting a calcium affinity which is dependent upon environmental conditions. As conditions change calmodulin binds either more or less calcium. Therefore polymers containing calmodulin and Ca2+ dye can be used as an environmental sensor. Many other enzymes affect concentrations of peroxide in an environment. The concentration of peroxide in the environment can affect the color of a dye (for example, by bleaching).
In another aspect, the present invention provides a process for preparing a sensor to detect the presence of at least one analyte as described above. The process comprises generally the steps of (a) incorporating into a polymer at least one enzyme that is selected to either (i) catalyze a reaction of the analyte to chemically convert the analyte to a product compound or (ii) be inhibited by the analyte in the presence of a substrate compound; and (b) incorporating into the polymer at least one indicator compound selected to produce a measurable change of state as a result of the interaction of the analyte and the enzyme.
In one embodiment, the enzyme and the indicator compound are incorporated into the polymer matrix during synthesis of the polymer. For example, polyurethane biosensor of the present invention above can be formed by mixing water, the enzyme, the indicator compound and an isocyanate functionalized polyurethane prepolymer. Incorporation of the enzyme(s) and indicator compound(s) into the polymer network
In still another aspect, the present invention provides a method for detecting the presence of at least one analyte. The method includes the step of exposing a sensor to an environment in which the analyte is present. As described above, the sensor includes at least one enzyme that is selected to either (i) catalyze a reaction of the analyte to chemically convert the analyte to a product compound or (ii) be inhibited by the analyte in the presence of a substrate compound. The sensor also includes at least one indicator compound selected to produce a measurable change of state as a result of the interaction of the analyte and the enzyme. Each of the enzyme and the indicator compound are incorporated within a single polymer. The method preferably further comprises the step of measuring the change of state (for example, viewing a color change).
In an additional aspect, the present invention provides a polymeric sensor operable to indicate the presence of an enzyme. The sensor includes at least one substrate compound and at least one indicator compound selected to produce a measurable change of state as a result of the interaction of the substrate and at least one target or analyte enzyme. As described for the sensors above, each of the indicator(s) and substrate(s) are incorporated within a single polymer. This sensor makes use of enzyme-substrate interactions to facilitate a change in the indicator compound as described above. The sensor can, for example, be used to detect the presence of enzymes using polymerized enzyme substrates.
In a further aspect, the present invention provides a process for preparing a sensor to detect the presence of at least one analyte enzyme comprising the steps of: incorporating at least one substrate compound that is selected to react in a reaction catalyzed by the analyte enzyme and incorporating into the polymer at least one indicator compound selected to produce a measurable change of state as a result of the interaction of the analyte enzyme and the substrate compound.
In still a further aspect, the present invention provides a method for detecting the presence of at least one analyte enzyme including the steps of: (a) exposing a sensor to an environment in which the analyte enzyme is present, the sensor including at least one substrate compound that is selected to react in a reaction catalyzed by the analyte enzyme and at least one indicator compound selected to produce a measurable change of state as a result of the interaction of the analyte enzyme and the substrate compound, each of the substrate compound and the indicator compound being incorporated within a single polymer; and (b) measuring the change of state.
In the case that the indicator compound(s) of the present invention undergoes a visible transition during enzyme catalysis of a target analyte or substrate, the need for cumbersome electrical equipment, electrodes, or special devices associated with prior enzymatic sensors is eliminated. In general, any compound that undergoes or produces an measurable transition in the presence of a reaction product is preferred for use in the present invention. Such compounds are referred to generally herein as xe2x80x9cdyes.xe2x80x9d As discussed above, the transition is preferably viewable by the xe2x80x9cnakedxe2x80x9d human eye.